A Colorimetric ELISA Enabling Quantification of APP Δ C31 in Cell Lysates and Cerebrospinal Fluid

INTRODUCTION

The amyloid precursor protein (APP) undergoes a variety of modifications through enzymatic cleavage leading to the formation of pro-Alzheimer’s disease (AD) and anti-AD biologically active peptides. One such cleavage product, APP δC31 results from the caspase cleavage of the full length APP protein (APP₆₉₅) at amino acid Asp664 as well as the concomitant formation of the 31 amino acid C-terminal peptide (C31). The APP δC31 truncated protein has been shown to accumulate in brain tissue of AD patients as well as in brain tissue from AD model mice and is suggested to play a role in the etiology of the disease. To enable the quantitative measurement of APP δC31 formation in cells and tissues we have developed a colorimetric ELISA assay. The assay is highly specific to APP δC31 exhibiting no cross-reactivity to APP. The assay has a sensitivity of 1 pM with an assay range of 12 to 1500 pM. The ELISA has been validated for use in cell lysates and cerebral spinal fluid and is indicated for use in serum and plasma. The applicability of the assay to drug screening studies in cell culture was demonstrated through simvastatin induction of APP δC31 production in the presence of increasing concentrations of a pan caspase inhibitor. The amount of APP δC31 in cell lysates decreased with increased concentration of caspase inhibitor. Establishing the ability of the assay to function in a wide variety of matrices provides the opportunity to evaluate APP δC31 as a biomarker for diagnosing and monitoring Alzheimer’s disease.

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